Urinary Proteome Profiling Using 2D-DIGE and LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 304

4. Notes

(1) If available the use of a dedicated clean room is highly
recommended. To prevent contamination by hair/skin
keratins gloves, aprons, facemask and hair caps need to
be worn while in the clean room.
(2) Sample preparation methods should remove salts, lipids,
detergents and nucleic acids as these can affect isoelectric
focusing.
(3) The buffers used during sample preparation need to be
compatible with the protein quantitation technique used.
(4) Cyanine dyes are water sensitive and need to be stored in
a water-free environment (silica gel may be used). DMF
used to make stock solution of cyanine dyes should be
99.5% pure and anhydrous. Resolubilisation of stock dyes
should be conducted under a nitrogen atmosphere.
(5) Adding 10% to these figures allows for variations in pipet-
ting and ensures sufficient sample for all replicates.
(6) The method detailed here is for minimal labelling using
cyanine dyes.
(7) The length of IPG gel strip, pH range and IEF gradient
can be optimised to achieve the best possible resolution
for the samples of interest.
(8) Prior to the second dimension separation and BEFORE
equilibration IEF gel strips can be stored at

80 ◦ Cfor

−

several weeks in a rigid container.
(9) The gels need to polymerise completely and should prefer-
ably be left overnight.
(10) Exceeding the stipulated times during reduction and alky-
lation of IPG gel strips may contribute to protein loss.
(11) Care should be taken not to introduce air bubbles at the
gel interface while inserting the IPG gel strip between the
glass plates. The overlaying agarose must solidify prior to
commencing second dimension separation.
(12) To ensure continuity use IPG gel strips from the same
batch and cast second dimension gels at the same time.
(13) Prior to scanning, both outer gel plate surfaces are wiped
clean and dry to ensure optimum scanned image quality.
(14) After applying GelCode Blue Safe Protein Stain gels do
not need to be destained to visualise proteins.
(15) GelCode Blue Safe Protein Stain gives very low back-
ground and gel does not

require destain; however,